Non-carboxylic Analogues of Naproxen: Design, Synthesis, and Pharmacological Evaluation of some...

Arch. Pharm. Chem. Life Sci. 2007, 340, 577 – 585 M. Amir et al. 577

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Non-carboxylic Analogues of Naproxen: Design, Synthesis,and Pharmacological Evaluation of some 1,3,4-Oxadiazole/Thiadiazole and 1,2,4-Triazole Derivatives

Mohammad Amir, Harish Kumar, and Sadique A. Javed

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi, India

A series of substituted 1,3,4-oxadiazole (2–7 and 14–19), 1,2,4-triazole (20–25), and 1,3,4-thiadia-zole (26–31) derivatives of naproxen have been synthesized by cyclization of 2-(6-methoxy-2-naphthyl)propanoic acid hydrazide 1 and N1[2-(6-methoxy-2-naphthyl) propanoyl]-N4-alkyl/aryl-thiosemicarbazides (8–13) under various reaction conditions. All the compounds were screenedfor their anti-inflammatory activity by carrageenan-induced rat paw edema test method. Com-pounds showing high anti-inflammatory activity were also tested for their analgesic, ulcero-genic, and lipid peroxidation. Few of the synthesized compounds showed significant anti-inflam-matory and analgesic activities along with reduced ulcerogenic effect and lipid peroxidation.

Keywords: Anti-inflammatory / Naproxen / 1,3,4-Oxadiazoles / Thiadiazoles / 1,2,4-Triazoles /

Received: March 27, 2007; accepted: July 24, 2007

DOI 10.1002/ardp.200700065

Introduction

The majority of non-steroidal anti-inflammatory drugs(NSAIDs) act via inhibition of cyclooxygenase thus pre-venting prostaglandin biosynthesis. However, this mech-anism of action is also responsible for their main undesir-able side effect, gastrointestinal (GI) ulceration and renalinjury [1]. The increase in hospitalization and deaths dueto GI-related disorders parallels the increased use ofNSAIDs [2]. Naproxen was one of the leading NSAIDs usedfor relieving arthritic pain, but its long-term use invitesGI complications ranging from stomach irritation to life-threatening GI ulceration and bleeding [3–5]. These clini-cal shortcomings comprise a major challenge confront-ing medicinal chemists to develop safer agents that spare

COX-1 and subsequently its gastric cytoprotective role.Recently, it was discovered that COX exist in two iso-forms, COX-1 and COX-2, which are regulated differently[6, 7]. COX-1 provides cytoprotection in the gastrointesti-nal tract whereas inducible COX-2 mediates inflamma-tion [8, 9]. Thus the discovery of COX-2 provided therationale for the development of drugs devoid of GI disor-ders while retaining clinical efficacy as anti-inflamma-tory agents. But the recent reports showed that selectiveCOX-2 inhibitors (coxibs) could lead to adverse cardiovas-cular effects [10]. Therefore, development of novel com-pounds having anti-inflammatory and analgesic activitywith an improved safety profile is still a necessity.

Synthetic approaches based upon NSAIDs chemicalmodification have been taken with the aim of improvingtheir safety profile. The literature survey revealed thatderivatization of the carboxylate function of NSAIDs alsoresulted in retained anti-inflammatory activity withreduced ulcerogenic potential [11–14]. Furthermore, ithas been reported in the literature that certain com-pounds bearing 1,3,4-oxadiazole/thiadiazole and 1,2,4-triazole nucleus possess significant anti-inflammatoryactivity [15–18]. In view of these observations and in con-tinuation of our research program on the synthesis of 5-membered heterocyclic compounds of aryl alkanoic acid

Correspondence: Dr. Mohammad Amir, Department of PharmaceuticalChemistry, Faculty of Pharmacy, Hamdard University, New Delhi-110062, India.E-mail: [email protected]: +91 11 260 59688, Extn. 5307

Abbreviations: carboxymethyl cellulose (CMC); gastrointestinal (GI);malondialdehyde (MDA); non-steroidal anti-inflammatory drugs(NSAIDs); serum glutamate oxaloacetate transaminase (SGOT); serumglutamate pyruvate transaminase (SGPT)

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578 M. Amir et al. Arch. Pharm. Chem. Life Sci. 2007, 340, 577 –585

derivatives [19–21], we report herein the synthesis ofsome newer, more potent analogues of 2-(6-methoxy-2-naphthyl)propanoic acid (naproxen) by cyclizing the car-boxylic group into 1,3,4-oxadiazole, 1,2,4-triazole, and1,3,4-thiadiazole nuclei, which have been found to pos-sess an interesting profile of anti-inflammatory and anal-gesic activity with significant reduction in their ulcero-genic effect.

Results and discussion

Chemistry2-(6-Methoxy-2-naphthyl)propanoic acid hydrazide 1 wasprepared by esterfication of 2-(6-methoxy-2-naphthyl)pro-panoic acid followed by treatment with hydrazinehydrate in absolute ethanol. Treatment of the hydrazide1 with appropriate aromatic acids in phosphorous oxy-chloride afforded 5-[1-(6-methoxy-2-naphthyl)ethyl]-2-sub-stituted-1,3,4-oxadiazoles 2–7. Furthermore, hydrazide 1on treatment with alkyl/aryl isothiocyanates gave N1[2-(6-methoxy-2-naphthyl)propanoyl]-N4-alkyl/aryl-thiosemi-carbazides 8–13. These thiosemicarbazides were oxida-tively cyclized to 5-[1-(6-methoxy-2-naphthyl)ethyl]-2-alkyl/aryl amino-1,3,4-oxadiazoles 14–19 by eliminationof H2S using iodine and potassium iodide in ethanolicsodium hydroxide. The thiosemicarbazides 8–13 on heat-

ing with 4N NaOH in ethanol underwent smooth cycliza-tion through dehydration to afford 4-alkyl/aryl-5-[1-(6-methoxy-2-naphthyl)ethyl]-3-mercapto-(4H)-1,2,4-tria-zoles 20–25. 5-[1-(6-Methoxy-2-naphthyl)ethyl]-2-alkyl/aryl amino-1,3,4-thiadiazoles 26–31 were obtained bycyclization of thiosemicarbazides 8–13 after treatmentwith cold concentrated sulphuric acid (Scheme 1). Thepurity of various synthesized compounds was checked byTLC and elemental analysis. Spectral data (1H-NMR, IR,and mass) of the synthesized compounds were in fullagreement with the proposed structures.

Biological studiesThe anti-inflammatory activity of the synthesized com-pounds 2–7, 14–29, and 31 was evaluated by carra-geenan-induced paw edema method of Winter et al. [23],edema being measured after 3 and 4 h of carrageenantreatment. Also, it was observed that compounds 7, 15,22, 25, and 28 showed anti-inflammatory activity 78.02%,74.99%, 75.75%, 76.51%, and 74.99% after 3 h, which wasfound to be greater than standard drug naproxen(74.23%). When the activity of these compounds wasmeasured after 4 h, only compound 22 showed activityequivalent to naproxen 81.81%. The rest of the com-pounds showed a lower activity. Since%-inhibition wasfound to be more after 4 h, this was made the basis of dis-cussion. All the derivatives of naproxen tested showed

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Scheme 1. Synthetic pathways to 1,3,4-oxadiazole (2–7 and 14-19), 1,2,4-triazole (20–25) and 1,3,4-thia-diazole (26–31) derivatives of naproxen.

Arch. Pharm. Chem. Life Sci. 2007, 340, 577 – 585 Azole Derivatives of Naproxen 579

anti-inflammatory activity ranging from 13.63 to 81.81%at an equimolar oral dose relative to 30 mg/kg naproxenafter 4 h, whereas the standard drug naproxen showed81.81% inhibition at the same oral dose (Table 2). The1,3,4-oxadiazole derivative of naproxen 7 having 1-(4-iso-butylphenyl)ethyl group at second position of the oxadia-zole ring showed anti-inflammatory activity (81.05%)almost equal to the standard drug naproxen. The replace-ment of this group by 2,4-dichlorophenoxy methyl 6 and4-chlorophenyl amino 15 groups resulted in a slightdecrease of activity (77.19% and 77.27%, respectively).The introduction of 2-chlorophenyl 4, 4-chlorophenyl 3


Table 1. Physical data of the synthesized compounds.

Com-pound

R Yield(%)

Mp.(8C)

Mol.Formula

Mol.Wt.

2 57 186 C21H18N2O2 330

3 76 198 C21H17ClN2O2 364

4 66 A300 C21H17ClN2O2 364

5 59 246 C21H16Cl2N2O2 399

6 68 178 C22H18Cl2N2O3 429

7 66 254 C27H30N2O2 414

8 58 150 C19H25N3O2S 359

9 67 148 C21H20ClN3O2S 413

10 69 144 C21H20BrN3O2S 458

11 57 162 C21H20FN3O2S 397

12 54 184 C22H23N3O2S 393

13 61 158 C23H25N3O2S 407

14 53 128 C19H23N3O2 325

15 61 154 C21H18ClN3O2 379

16 60 A300 C21H18BrN3O2 424

17 55 174 C21H18FN3O2 363

18 51 A300 C22H21N3O2 359

19 54 136 C23H23N3O2 373

Table 1. Continued.

Com-pound

R Yield(%)

Mp.(8C)

Mol.Formula

Mol.Wt.

20 76 176 C19H23N3OS 341

21 71 124 C21H18ClN3OS 396

22 70 210 C21H18BrN3OS 440

23 69 144 C21H18FN3OS 379

24 66 220 C22H21N3OS 375

25 67 186 C23H23N3OS 389

26 61 122 C19H23N3OS 341

27 59 170 C21H18ClN3OS 396

28 64 240 C21H18BrN3OS 440

29 57 170 C21H18FN3OS 379

30 61 252 C22H21N3OS 375

31 66 216 C23H23N3OS 389

Satisfactory analysis for CHN was obtained for all the com-pounds with in l0.3% of the theoretical values.


and 2,4-dichlorophenyl 5 groups at position two of theoxadiazole ring resulted in further decrease of activity(62.11%, 55.29%, and 51.51%, respectively). Replacementof these groups by phenyl 2 and 4-bromophenyl amino16 groups resulted in sharp decrease of activity (29.54%and 13.63%, respectively).

1,2,4-Triazole derivatives of naproxen showed anti-inflammatory activity ranging from 18.94 to 81.81%. Themaximum reduction in paw edema 81.81% (equal to nap-roxen) was shown by compound 22 having a 4-bromo-phenyl group at position four of triazole ring. When thisgroup was replaced by the 2-methylphenyl group 24, asharp decrease in activity (18.94%) was observed, whereasreplacement by 2,4-dimethylphenyl 25, resulted in anonly slight decrease in activity (79.54%). The other tria-zole derivatives showed weak anti-inflammatory activity.The cyclization of the carboxylic group of naproxen intothe thiadiazole ring also showed good anti-inflammatoryactivity ranging from 24.23% to 78.02%. Compound 28having a 4-bromophenyl amino group at position two ofthe thiadiazole ring showed 78.02% inhibition in rat pawedema. The activity was found to be slightly decreased(74.99%) when this group was replaced by a n-butylaminogroup 26. The other compounds showed moderate toweak anti-inflammatory activity.

The compounds which showed A70% anti-inflamma-tory activity were further tested for their analgesic activ-ity at the same oral dose as the one used for the anti-inflammatory activity. The analgesic activity was carriedout by the tail immersion method [24] and results are pre-sented as%-analgesia in Table 3. The analgesic activity ofcompounds 6, 7, 15, 22, 25, 26, and 28 was found to be inthe range from 20.8 to 86.6%. It was interesting to note

that the triazole derivative of naproxen 22, having a 4-bromophenyl group at the 4th position of triazole ring,having maximum anti-inflammatory activity alsoshowed maximum analgesic effect 86.6%, which is morethan the standard drug naproxen (73.5%). The oxadiazolederivatives 6 and 15 showed good analgesic activity(58.7% and 57.6%, respectively). The rest of the com-pounds showed moderate to weak analgesic activity.

The compounds, which were screened for their analge-sic effect, were further tested for their acute ulcerogenic-ity at an equimolar oral dose relative to 90 mg/kg nap-roxen. The ulcerogenic activity was carried out by themethod of Ciolli et al. [25]. Results showed that all thetested compounds exhibited reduction in the severityindex (Table 3) in comparison to the standard drug nap-roxen (severity index 2.250 l 0.11). The 1,3,4-thiadiazolederivative 28 having 4-bromophenyl amino group at the2nd position of the thiadiazole ring showed minimumulcerogenicity (severity index 0.417 l 0.08), whereas tria-zole derivative 25 and thiadiazole derivative 26 showedmaximum severity index (0.833 l 0.17 and 0.833 l 0.25,respectively). It was noted that triazole derivative 22which showed maximum anti-inflammatory and analge-sic activity also showed a very low severity index of 0.583l 0.08 as compared to naproxen. In general, the testedcompounds showed a better GI safety profile comparedto the reference drug.

All the compounds screened for ulcerogenic activitywere also analyzed for lipid peroxidation [26]. Lipid per-oxidation was measured as nanomol of malondialdehyde(MDA)/100 mg of gastric mucosa tissue. It has beenreported in the literature that compounds showing lessulcerogenic activity also showed reduced MDA content, a


Table 2. Anti-inflammatory activity of the synthesized compounds.

Compound Anti-inflammatory activity %inhibition lSEMa)

Compound Anti-inflammatory activity %inhibition lSEMa)

After 3h After 4 h After 3 h After 4 h

2 26.51 l 3.60 29.54 l 3.05b) 19 47.72 l 1.95 52.28 l 2.81b)

3 53.78 l 3.60 55.29 l 3.97b) 20 18.93 l 3.20 23.47 l 2.47b)

4 62.11 l 2.80 62.11 l 2.80b) 21 52.26 l 2.56 55.29 l 2.47b)

5 49.24 l 1.40 51.51 l 0.96b) 22 75.75 l 2.25 81.81 l 2.046 73.48 l 2.16 77.19 l 2.37 23 33.33 l 4.18 36.36 l 4.84b)

7 78.02 l 3.60 81.05 l 3.60 24 17.42 l 4.30 18.94 l 3.79b)

14 45.45 l 3.52 46.96 l 3.45b) 25 76.51 l 2.17 79.54 l 1.9515 74.99 l 3.27 77.27 l 2.35 26 71.20 l 2.53 74.99 l 1.9416 11.36 l 1.94 13.63 l 1.66b) 27 29.54 l 3.85 32.57 l 3.97b)

17 44.69 l 3.60 49.24 l 3.20b) 28 74.99 l 2.56 78.02 l 1.8218 52.27 l 2.81 55.30 l 2.73b) 29 20.45 l 3.47 24.23 l 3.65b)

Naproxen 74.23 l 3.03 81.81 l 2.65 31 38.63 l 4.02 41.66 l 4.30b)

a) Relative to the standard; the data was analyzed by ANOVA followed by dunnett's multiple comparision test for n = 6.b) p a 0.01.


byproduct of lipid peroxidation. Naproxen (standarddrug) showed maximum lipid peroxidation (9.04 l 0.24)whereas the control group showed 3.25 l 0.05. It wasfound that all cyclized derivatives showing less ulcero-genic activity also showed reduction in lipid peroxida-tion Table 3. Thus, these studies demonstrate that thesynthesized compounds have inhibited the induction ofgastric mucosal lesion.

The 1,3,4-oxadiazole, 1,2,4-triazole, and 1,3,4-thiadia-zole derivatives of naproxen 7, 22, and 28, respectively,showing potent anti-inflammatory activity with reducedulcerogenicity and lipid peroxidation were further stud-ied for their hepatotoxic effect. All the compounds werestudied for their effect on biochemical parameters(serum enzymes, total protein, and total albumin) [27–29] and histopathology of liver [30]. As shown in Table 4,activities of the liver enzymes serum glutamate oxaloace-tate transaminase (SGOT) and serum glutamate pyruvatetransaminase (SGPT), alkaline phosphatase, and total pro-tein, total albumin almost remain the same with respectto the control values. The histopathological studies of the

liver samples do not show any significant pathologicalchanges in comparison to the control group (Fig. 1). Nohepatocyte necrosis or degeneration was seen in any ofthe samples.

In conclusion, various oxadiazole, thiadiazole, and tri-azole derivatives of naproxen were prepared with theobjective of developing better anti-inflammatory mole-cules with minimum ulcerogenic activity. It was interest-ing to note that seven cyclized compounds 6, 7, 15, 22, 25,26, and 28 were found to have significant anti-inflamma-tory activity. When these compounds were subjected toanalgesic activity by the tail immersion method in mice,except 25, all compounds showed significant activity.Compound 22 was found to have a higher analgesic activ-ity (86.6%) than the standard drug naproxen (75.5%).These compounds were also tested for ulcerogenic activ-ity and showed a significant reduction in the severityindex compared to the standard reference drug. Fromthese studies, compound 22, 4-(4-bromophenyl)-5-[1-(6-methoxy-2-naphthyl)ethyl]-3-mercapto-(4H)-1,2,4-triazolehas emerged as the lead compound, which showed maxi-


Table 3. Analgesic, ulcerogenic, and lipid peroxidation activity of selected compounds.

Compound Analgesic activitya) Ulcerogenic activity(Severity index lSEM)d)

nmol MDA contentlSEM/100 mg tissued)

Pre-treatment normal0 h (s)

Post-treatment after4 h (s)

% Inhibition

6 1.05 l 0.070 1.67 l 0.113c) 58.7 0.750 l 0.17e) 5.53 l 0.34e)

7 1.11 l 0.119 1.66 l 0.197c) 50.2 0.583 l 0.08e) 5.45 l 0.47e)

15 1.25 l 0.208 1.97 l 0.193b) 57.6 0.500 l 0.00e) 4.78 l 0.21e)

22 1.28 l 0.149 2.40 l 0.154b) 86.6 0.583 l 0.08e) 5.43 l 0.45e)

25 1.54 l 0.164 1.86 l 0.166c) 20.8 0.833 l 0.17e) 5.54 l 0.37e)

26 1.46 l 0.078 2.07 l 0.077 41.8 0.833 l 0.25e) 5.80 l 0.46e)

28 1.28 l 0.157 1.91 l 0.059 48.9 0.417 l 0.08e) 4.70 l 0.29e)

Naproxen 1.17 l 0.086 2.03 l 0.039b) 73.5 2.250 l 0.11 9.04 l 0.24Control – – – 0.00 3.25 l 0.05

a) Relative to normal; the data was analyzed by paired student's t-test for n = 6.b)p a 0.0001.c)p a 0.005.d) Relative to standard; the data was analyzed by ANOVA followed by dunnett's multiple comparision test for n = 6.e) p a 0.01.

Table 4. Effect of compounds on serum enzymes, total protein, and total albumin.

Compound SGOT Units/mla) SGPT Units/mla) AlkalinePhophatasea)

Total protein(g/dL)a)

Total albumin(g/dL)a)

Control 148.67 l 1.50 27.67 l 0.84 13.06 l 0.25 1.80 l 0.01 1.67 l 0.017 142.17 l 0.98b) 25.33 l 0.61 18.69 l 0.16b) 1.82 l 0.05 1.74 l 0.06

22 129.33 l 0.76b) 18.83 l 0.60b) 14.85 l 0.14b) 1.89 l 0.12 1.74 l 0.1328 139.67 l 1.65b) 23.17 l 0.75b) 14.25 l 0.14b) 1.59 l 0.05 1.44 l 0.05

a) Relative to control; the data was analyzed by ANOVA followed by dunnett's multiple comparison test, for n = 6.b) p a 0.01.


mum anti-inflammatory and analgesic effects along withreduction in ulcerogenic potential and lipid peroxida-tion Thus the series provided new opportunities for possi-ble modification of pharmacophoric requirements andfuture exploitations.

The authors are thankful to the Head of the Department Phar-maceutical Chemistry, for providing laboratory facilities andto the Central Drug Research Institute (CDRI) for spectral anal-ysis of the compounds. The authors are also thankful to Mrs.Shaukat Shah, in charge of the animal house, for providingWistar rats, and Dr. A. Mukherjee, MD, Department of Pathol-

ogy, All India Institute of Medical Sciences (AIIMS), New Delhi,for carrying out the histopathological studies.

Experimental

Melting points were determined in open capillary tubes and areuncorrected. IR (KBr) spectra were recorded on a Nicolet 5PCFTIR spectrometer (mmax in cm – 1) (Nicolet, Madison, WI, USA) and1H-NMR spectra were recorded in CDCl3/DMSO-d6 on a BrukerDRX-300 (300 MHz FT NMR) spectrometer (Bruker Bioscience,Billerica, MA, USA) using TMS as internal reference (chemicalshift in d ppm). Mass spectra were recorded at Jeol SX-102 (FAB)


Figure 1. Histopathological studies of the liver.


spectrometer (Jeol, Tokyo, Japan). Chemicals were purchasedfrom Merck Chemical Company,Gibbstown, NJ, USA), S. D. Fine(India) and Qualigens (India). Ethyl-2-(6-methoxy-2-naphthyl)pro-panoate was prepared by the procedure given in the literature[22].

Chemistry

2-(6-Methoxy-2-naphthyl)propanoic acid hydrazide 1To a mixture of ethyl-2-(6-methoxy-2-naphthyl)propanoate(0.01 mol) and hydrazine hydrate (0.05 mol), absolute ethanol(50 mL) was added and it was refluxed for 24 h on a water bath.The mixture was concentrated, cooled, and poured into crushedice. It was kept for 4 –5 h at room temperature and the solidmass separated out was filtered, dried, and recrystallized fromethanol. Mp 948C; Yield 61%; 1H-NMR (CDCl3): 1.60 (d, J = 7.1 Hz,3H, CH3); 3.66 (q, J = 7.1 Hz, 1H, CH); 3.92 (s, 3H, OCH3); 6.70 (s,2H, NH2); 7.13 –7.79 (m, 7H, 6-ArH and CONH).

General procedure for the synthesis of 5-[1-(6-methoxy-2-naphthyl)ethyl]-2-substituted-1,3,4-oxadiazoles 2–72-(6-Methoxy-2-naphthyl)propanoic acid hydrazide 1 (0.001 mol)and the appropriate aromatic acid (0.001 mol) were dissolved inphosphorus oxychloride and refluxed for 4–6 h. The reactionwas slowly poured over crushed ice and kept overnight. Solidthus separated out was filtered, treated with dilute NaOH,washed with water, and recrystallized with ethanol.

The IR spectra of the compounds 2–7 showed bands at 2953 –2929 (C-H); 1623 –1600 (C=N) cm – 1.

1H-NMR (CDCl3) 5: 1.67 (d, J = 7.0 Hz, 3H, CH3); 3.55 (s, 3H,OCH3); 3.92 (q, J = 7.0 Hz, 1H, CH); 7.13–8.09 (m, 9H, ArH). Massspectra of the compound exhibited the molecular ion peak at m/z 399 [M+], other important fragments were found at m/z 401[M++2], 253, 213, 185, 171.

1H-NMR (DMSO-d6) 6: 1.48 (d, J = 7.0 Hz, 3H, CH3); 3.87 –3.93 (m,4H, CH and OCH3); 4.70 (s, 2H, OCH2); 7.14 –7.78 (m, 9H, ArH).Mass spectra of the compound exhibited the molecular ion peakat m/z 429 [M+], other important fragments were found at m/z431 [M++2], 253, 213, 185, 171.

1H-NMR (DMSO-d6) 7: 0.97 [d, J = 6.5 Hz, 6H, (CH3)2]; 1.43 (d, J =7.0 Hz, 3H, CH3); 1.52 (d, J = 7.0 Hz, 3H, CH3); 1.84-1.92 (m, 1H,CHCH2); 2.47 (d, J = 7.0 Hz, 2H, CH2); 3.81-3.86 (m, 4H, CH &OCH3); 3.94 (q, J = 7.0 Hz, 1H, CHCH3); 6.87–7.92 (m, 10H, ArH).Mass spectra of the compound exhibited the molecular ion peakat m/z 414 [M+], other important fragments were found at m/z253, 213, 185, 171.

General procedure for the synthesis of N1[2-(6-methoxy-2-naphthyl)propanoyl]-N4-alkyl/aryl-thiosemicarbazides8–13A mixture of 2-(6-methoxy-2-naphthyl)propanoic acid hydrazide1 (0.10 mol), alkyl/aryl isothiocyanate (0.10 mol) and ethanol(50 mL) was refluxed for 2–8 h on a water bath. It was then con-centrated, cooled, and kept overnight in the refrigerator. Thesolid separated out was filtered, dried, and recrystallized with asuitable solvent. The IR spectra of the compounds 8–13 showedbands at 3316–3285 (N-H); 2955 –2933 (C-H); 1683–1674 (C=O);1108-1033 (C=S) cm – 1.

1H-NMR (CDCl3) 10: 1.47 (d, J = 6.8 Hz, 3H, CH3); 3.86–3.94 (m,4H, CH and OCH3); 7.13–7.79 (m, 10H, ArH); 9.60 (bs, 1H, ArNH);9.71 (bs, 1H, CSNH); 10.17 (bs, 1H, CONH). Mass spectra of the

compound exhibited the molecular ion peak at m/z 458 [M+],other important fragments were found at m/z 460 [M++2], 378,245, 213, 185, 171.

1H-NMR (CDCl3) 11: 1.51 (d, J = 6.8 Hz, 3H, CH3); 3.82–3.90 (m,4H, CH and OCH3); 7.01–7.90 (m, 10H, ArH); 9.65 (bs, 1H, ArNH);9.87 (bs, 1H, CSNH); 10.31 (bs, 1H, CONH).

1H-NMR (CDCl3) 12: 1.39 (d, J = 6.8 Hz, 3H, CH3); 1.94 (s, 3H, o-CH3); 3.60 –3.70 (m, 4H, CH and OCH3); 6.79 –7.56 (m, 10H, ArH);8.36 (bs, 1H, ArNH); 9.03 (bs, 1H, CSNH); 9.42 (bs, 1H, CONH).Mass spectra of the compound exhibited the molecular ion peakat m/z 393 [M+], other important fragments were found at m/z243, 213, 185, 171.

General procedure for the synthesis of 5-[1-(6-methoxy-2-naphthyl)ethyl]-2-alkly/arylamino-1,3,4-oxadiazoles 14–19A suspension of 8–13 (0.002 mol) in ethanol (50 mL) was dis-solved in aqueous sodium hydroxide (5N) with cooling and stir-ring resulting in the formation of a clear solution. To this, iodinein potassium iodide solution (5%) was added dropwise with stir-ring till the color of iodine persisted at room temperature. Thereaction mixture was then refluxed for 3 –5 h on a water bath. Itwas then concentrated, cooled, and the solid separated out wasfiltered, dried, and recrystallized with ethanol. The IR spectra ofthe compounds 14–19 showed bands at 3310 –3190 (N-H);2954 –2929 (C-H); 1653 –1612 (C=N) cm – 1.

1H-NMR (CDCl3) 14: 0.71 (t, J = 6.9 Hz, 3H, CH3); 1.16 –1.22 (m,2H, CH3CH2); 1.55 –1.62 (m, 2H, CH3CH2CH2); 1.73 (d, J = 6.9 Hz,3H, CHCH3); 3.65 (t, J = 6.9 Hz, 2H, NCH2); 3.91 (s, 3H, OCH3); 4.15(q, J = 6.9 Hz, 1H, CH); 7.12 –7.70 (m, 7H, 6 ArH and NH). Massspectra of the compound exhibited the molecular ion peak at m/z 325 [M+], other important fragments were found at m/z 310,253, 213, 185, 171.

1H-NMR (CDCl3) 15: 1.80 (d, J = 7.0 Hz, 3H, CH3); 3.90 (s, 3H,OCH3); 4.36 (q, J = 7.0 Hz, 1H, CH); 7.02 –7.73 (m, 10H, ArH); 9.88(bs, 1H, NH). 1H-NMR (CDCl3) 17: 1.80 (d, J = 6.9 Hz, 3H, CH3); 3.91(s, 3H, OCH3); 4.36 (q, J = 6.9 Hz, 1H, CH); 6.93 –7.72 (m, 10H,ArH); 9.50 (bs, 1H, NH). Mass spectra of the compound exhibitedthe molecular ion peak at m/z 363 (M+), other important frag-ments were found at m/z 364 [M++1], 365 [M++2], 344, 253, 213,185, 171.

1H-NMR (CDCl3) 18: 1.79 (d, J = 7.0 Hz, 3H, CH3); 2.17 (s, 3H, o-CH3); 3.91 (s, 3H, OCH3); 4.41 (q, J = 7.0 Hz, 1H, CH); 7.07 –7.81 (m,10H, ArH); 8.03 (bs, 1H, NH).

1H-NMR (CDCl3) 19: 1.79 (d, J = 7.1 Hz, 3H, CH3); 2.20 (s, 3H, o-CH3); 2.29 (s, 3H, p-CH3); 3.91 (s, 3H, OCH3); 4.36 (q, J = 7.1 Hz, 1H,CH); 6.96–7.73 (m, 9H, ArH); 8.03 (bs, 1H, NH). Mass spectra ofthe compound exhibited the molecular ion peak at m/z 373 [M+],other important fragments were found at m/z 374 [M++1], 358,344, 253, 213, 185, 171.

General procedure for the synthesis of 4-alkyl/aryl-5-[1-(6-methoxy-2-naphthyl)ethyl]-3-mercapto-(4H)-1,2,4-triazoles 20–25A suspension of 8–13 (0.002 mol) in ethanol (50 mL) was dis-solved in aqueous sodium hydroxide (4N), resulting in the for-mation of a clear solution. The reaction mixture was refluxedfor 4–6 h on a water bath, concentrated, cooled, and filtered.The pH of the filtrate was adjusted between 5–6 with acetic acidand kept aside for 1–2 h. The solid separated out was filtered,washed with water, dried, and recrystallized with ethanol. The



IR spectra of the compounds 20–25 showed bands at 2950–2918(C-H); 2786–2715 (S-H); 1638–1604 (C=N) cm – 1.

1H-NMR (CDCl3) 20: 0.72 (t, J = 6.9 Hz, 3H, CH3); 1.16 –1.25 (m,4H, CH3CH2CH2); 1.72 (d, J = 6.9 Hz, 3H, CHCH3); 3.81-3.92 (m, 4H,CH and OCH3); 4.15 (t, J = 6.9 Hz, 2H, NCH2); 7.12 –7.73 (m, 6H,ArH); 10.83 (bs, 1H, SH). Mass spectra of the compound exhibitedthe molecular ion peak at m/z 341 [M+], other important frag-ments were found at m/z 342 [M++1], 308, 285, 282, 185, 171.

1H-NMR (CDCl3) 21: 1.67 (d, J = 7.0 Hz, 3H, CH3); 3.81–3.92 (m,4H, CH & OCH3); 6.81 –7.61 (m, 10H, ArH); 11.79 (bs, 1H, SH).Mass spectra of the compound exhibited the molecular ion peakat m/z 396 [M+], other important fragments were found at m/z398 [M++2], 363, 328, 302, 185, 171.

1H-NMR (CDCl3) 22: 1.67 (d, J = 6.8 Hz, 3H, CH3); 3.84–3.92 (m,4H, CH & OCH3); 6.74 –7.62 (m, 10H, ArH); 11.27 (bs, 1H, SH).Mass spectra of the compound exhibited the molecular ion peakat m/z 440 [M+], other important fragments were found at m/z441 [M++1], 442 [M++2], 407, 327, 301, 185, 171.

1H-NMR (CDCl3) 23: 1.67 (d, J = 7.0 Hz, 3H, CH3); 3.87–3.92 (m,4H, CH and OCH3); 6.97–7.61 (m, 10H, ArH); 11.99 (bs, 1H, SH).

1H-NMR (CDCl3) 24: 1.69 (d, J = 7.1 Hz, 3H, CH3); 1.97 (s, 3H,CH3); 3.84-3.90 (m, 4H, CH & OCH3); 6.88 –7.82 (m, 10H, ArH);10.90 (bs, 1H, SH). Mass spectra of the compound exhibited themolecular ion peak at m/z 375 [M+], other important fragmentswere found at m/z 342, 316, 302, 185, 171.

1H-NMR (CDCl3) 25: 1.69 (d, J = 7.0 Hz, 3H, CH3); 2.14 (s, 3H, o-CH3); 2.43 (s, 3H, p-CH3); 3.79 –3.91 (m, 4H, CH and OCH3); 6.79 –7.68 (m, 9H, ArH); 11.62 (bs, 1H, SH). Mass spectra ofthe com-pound exhibited the molecular ion peak at m/z 389 [M+], otherimportant fragments were found at m/z 356, 330, 185, 171.

General procedure for the synthesis of 5-[1-(6-methoxy-2-naphthyl)ethyl]-2-alkyl/aryl amino-1,3,4-thiadiazoles26–31The thiosemicarbazide 8–13 (0.001 mol) was added graduallywith stirring to cooled conc. sulphuric acid (10 mL) during10 min. The mixture was further stirred for another 5 h in an icebath. It was then poured over crushed ice with stirring. The solidseparated out was filtered, washed with water, dried, and recrys-tallized with ethanol. The IR spectra of the compounds 26–31showed bands at 3369–3186 (N-H); 2934 –2900 (C-H); 1626 –1606(C=N) cm – 1.

1H-NMR (DMSO-d6) 26: 0.84 (t, J = 7.0 Hz, 3H, CH3); 1.30 –1.35(m, 2H, CH3CH2); 1.53 –1.57 (m, 2H, CH3CH2CH2); 1.68 (d, J =7.0 Hz, 3H, CHCH3); 3.28 (t, J = 6.9 Hz, 2H, NCH2); 3.83 (s, 3H,OCH3); 4.59 (q, J = 7.0 Hz, 1H, CH); 7.19 –7.85 (m, 6H, ArH); 8.22(bs, 1H, NH).

1H-NMR (CDCl3) 27: 1.82 (d, J = 7.0 Hz, 3H, CH3); 3.91 (s, 3H,OCH3); 4.53 (q, J = 7.0 Hz, 1H, CH); 7.13 –7.71 (m, 10H, ArH); 8.27(bs, 1H, NH). 1H-NMR (DMSO-d6) 28: 1.72 (d, J = 6.5 Hz, 3H, CH3);3.83 (s, 3H, OCH3); 4.63 (q, J = 7.0 Hz, 1H, CH); 7.16–7.68 (m, 10H,ArH); 8.21 (bs, 1H, NH). Mass spectra of the compound exhibitedthe molecular ion peak at m/z 440 [M+], other important frag-ments were found at m/z 442 [M++2], 362, 243, 255, 185.

1H-NMR (DMSO-d6) 31: 1.72 (d, J = 7.0 Hz, 3H, CH3); 2.16 (s, 3H, o-CH3); 2.23 (s, 3H, p-CH3); 3.82 (s, 3H, OCH3); 4.50 (q, J = 7.0 Hz, 1H,CH); 7.05 –7.87 (m, 9H, ArH); 8.21 (bs, 1H, NH).

Biological evaluationAdult male Wistar strain rats of either sex, weighing 150 –200 gwere used. The animals were allowed food and water ad libitum.

They were housed in a room at 25 l 28C, and 50 l 5% relativehumidity with 12 h light/dark cycle. The animals were randomlyallocated into groups at the beginning of all the experiments.All the test compounds and the reference drugs were adminis-tered orally, suspended in 0.5% carboxymethyl cellulose (CMC)solution.

Anti-inflammatory activityThe synthesized compounds were evaluated for their anti-inflam-matory activity using carrageenan-induced hind paw edemamethod of Winter et al. [23]. The animals were randomly allocatedinto groups of six animals each. One group was kept as controland received only 0.5% CMC solution. Group II was kept as stand-ard and received naproxen (30 mg/kg, p. o.). Carrageenan solution(0.1% in sterile 0.9% NaCl solution) in a volume of 0.1 mL wasinjected subcutaneously into the sub-plantar region of the righthind paw of each rat 1 h after the administration of the test com-pounds and standard drugs. The right hind paw volume wasmeasured before and after 3 and 4 h of carrageenan treatment bymeans of a plethysmometer. The percent anti-inflammatoryactivity was calculated according to the following formula.

Percent anti-inflammatory activity = (Vc – Vt/Vc)6100

where Vt represents the mean increase in paw volume in ratstreated with test compounds and Vc represents the meanincrease in paw volume in the control group of rats.

Analgesic activityAnalgesic activity was evaluated by the tail immersion method[24]. Swiss albino mice allocated into different groups consistingof six animals in each of either sex, weighing 25 –30 g were usedfor the experiment. Analgesic activity was evaluated after oraladministration of the test compounds at an equimolar dose rela-tive to 30 mg/kg naproxen. Test compounds and standard drugswere administered orally as suspension in CMC solution inwater (0.5% w/v). The analgesic activity was assessed before andafter an 4 h interval of administration of the test compoundsand standard drugs. The lower 5 cm portion of the tail was gen-tly immersed into thermostatically controlled water at 55 l0.58C. The time (in seconds) for tail withdrawal from the waterwas taken as the reaction time with a cut-off time of immersion,set at 10 s for both controls as well as treated animals.

Acute ulcerogenicityAcute ulcerogenicity was determined according to Cioli et al.[25]. The animals were allocated into different groups consistingof six animals in each group. Ulcerogenic activity was evaluatedafter oral administration of the test compounds at an equimolardose relative to 90 mg/kg naproxen. Control group received only0.5% CMC solution. Food but not water was removed 24 h beforeadministration of the test compounds. After the drug treatment,the rats were fed with normal a diet for 17 h and then sacrificed.The stomach was removed and opened along the greater curva-ture, washed with distilled water, and cleaned gently by dippingin normal saline. The mucosal damage was examined by meansof a magnifying glass. For each stomach the mucosal damagewas assessed according to the following scoring system:

0.5: redness; 1.0: spot ulcers; 1.5: hemorrhagic streaks; 2.0:ulcers A3 but F5; 3.0: ulcers A5. The mean score of each treatedgroup minus the mean score of the control group was regardedas severity index of the gastric mucosal damage.



Lipid peroxidationLipid peroxidation in the gastric mucosa was determinedaccording to the method of Ohkawa et al. [26]. After screeningfor ulcerogenic activity, the gastric mucosa was scraped withtwo glass slides, weighed (100 mg) and homogenized in 1.8 mLof 1.15% ice cold KCl solution. The homogenate was supple-mented with 0.2 mL of 8.1% sodium dodecyl sulphate (SDS),1.5 mL of acetate buffer (pH 3.5), and 1.5 mL of 0.8% thiobarbitu-ric acid (TBA). The mixture was heated at 958C for 60 min. Aftercooling, the reactants were supplemented with 5 mL of a mix-ture of n-butanol and pyridine (15 : 1 v/v), shaken vigorously for1 min and centrifuged for 10 min at 4000 rpm. The supernatantorganic layer was taken out and absorbance was measured at532 nm in an UV spectrophotometer. The results were expressedas nmol MDA/100 mg tissue, using the extinction coefficient1.566105 cm – 1 M – 1.

Hepatotoxic studiesThe study was carried out with Wistar albino rats of either sexweighing 150 –200 g. The animals were divided into fourgroups, six rats in each. Group I was kept as control and receivesonly vehicle (0.5% w/v solution of carboxymethylcellulose inwater), the other groups received test compounds at an equimo-lar oral dose relative to 30 mg/kg naproxen in 0.5% w/v solutionof CMC in water once in a day for 15 days. After the treatment(15 days) blood was obtained from all the groups of rats by punc-turing the retro-orbital plexus. Blood samples were allowed toclot for 45 min at room temperature and serum was separatedby centrifugation at 2500 rpm for 15 min and analyzed for vari-ous biochemical parameters.

Assessment of liver functionAssessment of liver function such as serum glutamate oxaloace-tate transaminase (SGOT) and serum glutamate pyruvate transa-minase (SGPT) were estimated by a reported method [27]. Thealkaline phosphatase, total protein, and total albumin weremeasured according to the reported procedures [28 –29]. All thedata are recorded in Table 4.

Histopathological studies of liverThe histopathological studies were carried out by reportedmethod [30]. The rats were sacrificed under light-ether anesthe-sia 24 h after the last dosage. The livers were removed andwashed with normal saline and stored in formalin solution. Sec-tions of 5–6 microns in thickness were cut, stained with haema-toxylin and eosin, and then studied under an electron micro-scope (Fig. 1).

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Non-carboxylic Analogues of Naproxen: Design, Synthesis, and Pharmacological Evaluation of some...

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